Abstract Despite the introduction of combination antiretroviral therapy (cART), central nervous system (CNS) disease remains a significant challenge in patients with HIV. The abuse of drugs such as heroin is associated with treatment non-compliance, greater risk of viral transmission, and more rapid clinical progression of HIV disease. The overarching hypothesis behind the present project is that the analysis of molecular profiles of neuronal and glia cells will reveal key genes that are dysregulated by HIV and by the interaction of HIV with heroin abuse, resulting in cognitive impairments and depression. Their identification will point to transformative new mechanistic hypotheses on neuroAIDS pathogenesis and, consequently, to novel therapeutic targets to improve neuropsychological functioning in people with HIV. To test the present hypothesis, we propose to use validated systems biology strategies for the reconstruction and interrogation of a genome-scale integrated gene regulatory network (interactome) using gene profiles by RNA sequencing (RNA-Seq) from HIV-1 transgenic (Tg) and wild-type rats in a state-of-the-art paradigm of voluntary intravenous drug self-administration under long access (LgA) conditions, which leads to dependent heroin intake, and short access (ShA) conditions, which is characterized by a non-dependent, more ?recreational? pattern of heroin use. Because of the pivotal roles of glia cells in the pathogenesis of neuronal dysfunction in the setting of neuroAIDS as well as in neuroinflammatory processes induced by drugs of abuse such as opiates, for the proposed interactome we will profile neurons, astrocytes and microglia purified by fluorescence-activated cell sorting (FACS) from key brain regions involved in the effects of HIV and drug dependence. We will interrogate the interactome to identify the gene network dysregulations driven by the interaction of HIV and dependent and non-dependent heroin intake. Specific mechanistic hypotheses derived from interactome reconstruction and interrogation will be tested with viral vector-mediated overexpression or down-regulation and genetically modified rats and mice in conjunction with additional rounds of gene expression profiling, behavioral analyses and neuropathology analyses to elucidate their contributions to the effects of HIV and heroin on cognition, depression-like behavior, and neurodegeneration. Central nervous system (CNS) disease remains a pressing problem in the HIV pandemic despite the introduction of cART. Injection drug use is associated with treatment non-compliance, greater risk of viral transmission, and more rapid clinical progression of HIV disease. The present proposal will bring to bear a systems biology approach to identify and test new mechanistic hypotheses that may lead to novel transformative therapeutic concepts to improve neuropsychological functioning in people with HIV.